Survival of Swiss-Webster mouse cerebellar granule neurons is promoted by a combination of potassium channel blockers

<p>Cultured cerebellar granule neurons (CGN) are commonly used to assess neurotoxicity, but are routinely maintained in supraphysiological (25 mM) extracellular K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> concentrations [K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup>]<sub style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">o</sub>. We investigated the effect of potassium channel blockade on survival of CGN derived from Swiss-Webster mice in supraphysiological (25 mM) and physiological (5.6 mM) [K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup>]<sub style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">o</sub>. CGN were cultured for 5 days in 25 mM K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup>, then in 5.6 mM K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> or 25 mM K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> (control). Viability, assayed 24 h later by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction and by lactate dehydrogenase (LDH) release, was ∼50% in 5.6 mM K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> versus 25 mM K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> (<em>p</em> &lt; .001). Potassium channel blockers, 2 mM 4-aminopyridine (4-AP), 2 mM tetraethylammonium (TEA) or 1 mM Ba<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">2+</sup>, individually afforded limited protection in 5.6 mM K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup>. However, survival in 5.6 mM K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> with a combination of 4-AP, TEA and Ba<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">2+</sup> was similar to survival in 25 mM K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> without blockers (<em>p</em> &lt; .001 versus 5.6 mM K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> alone). CGN survival in 25 mM K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> was attenuated 25% by 2 μM nifedipine (<em>p</em> &gt; .001), but nifedipine did not attenuate neuroprotection by K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> channel blockers. Together, these results suggest that the survival of CGN depends on the K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> permeability of the membrane rather than the activity of a particular type of K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> channel, and that the mechanism of neuroprotection by K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup> channel blockers is different from that of elevated [K<sup style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">+</sup>]<sub style="border: 0px; font-size: 0.75em; margin: 0px; padding: 0px; line-height: 0; color: rgb(46, 46, 46); font-family: 'Arial Unicode MS', 'Arial Unicode', Arial, 'URW Gothic L', Helvetica, Tahoma, sans-serif; text-align: justify; word-spacing: -1.03587377071381px; background-color: rgb(255, 255, 255);">o</sub>.</p>

Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

Low SNR Capacity for MIMO Rician and Rayleigh-Product Fading Channels with Single Co-channel Interferer and Noise

This paper studies the ergodic capacity of multiple-input multiple-output (MIMO) systems with a single co-channel interferer in the low signal-to-noise-ratio (SNR) regime. Two MIMO models namely Rician and Rayleigh-product channels are investigated. Exact analytical expressions for the minimum energy per information bit, Eb/N0min, and wideband slope, S0, are derived for both channels. Our results show that the minimum energy per information bit is the same for both channels while their wideband slopes differ significantly. Further, the impact of the numbers of transmit and receive antennas, the Rician K factor, the channel mean matrix and the interference-to-noise-ratio (INR) on the capacity, is addressed. Results indicate that interference degrades the capacity by increasing the required minimum energy per information bit and reducing the wideband slope. Simulation results validate our analytical results.

Ion channel gates: comparative analysis of energy barriers.

The energetic profile of an ion translated along the axis of an ion channel should reveal whether the structure corresponds to a functionally open or closed state of the channel. In this study, we explore the combined use of Poisson–Boltzmann electrostatic calculations and evaluation of van der Waals interactions between ion and pore to provide an initial appraisal of the gating state of a channel. This approach is exemplified by its application to the bacterial inward rectifier potassium channel KirBac3.1, where it reveals the closed gate to be formed by a ring of leucine (L124) side chains. We have extended this analysis to a comparative survey of gating profiles, including model hydrophobic nanopores, the nicotinic acetylcholine receptor, and a number of potassium channel structures and models. This enables us to identify three gating regimes, and to show the limitation of this computationally inexpensive method. For a (closed) gate radius of 0.4 nm

Short Isoforms of the Cold Receptor TRPM8 Inhibit Channel Gating by Mimicking Heat Action Rather than Chemical Inhibitors

Transient receptor potential (TRP) channels couple various environmental factors to changes in membrane potential, calcium influx, and cell signaling. They also integrate multiple stimuli through their typically polymodal activation. Thus, although the TRPM8 channel has been extensively investigated as the major neuronal cold sensor, it is also regulated by various chemicals, as well as by several short channel isoforms. Mechanistic understanding of such complex regulation is facilitated by quantitative single-channel analysis. We have recently proposed a single-channel mechanism of TRPM8 regulation by voltage and temperature. Using this gating mechanism, we now investigate TRPM8 inhibition in cell-attached patches using HEK293 cells expressing TRPM8 alone or coexpressed with its short sM8-6 isoform. This is compared with inhibition by the chemicals N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC) and clotrimazole or by elevated temperature. We found that within the seven-state single-channel gating mechanism, inhibition of TRPM8 by short sM8-6 isoforms closely resembles inhibition by increased temperature. In contrast, inhibition by BCTC and that by clotrimazole share a different set of common features. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis:in vitro evidence in cultured epithelial cells

BACKGROUND:<br/>Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR.<br/>OBJECTIVES:<br/>The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects.<br/>METHODS:<br/>Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function.<br/>RESULTS:<br/>Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P &lt; 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P &lt; 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway.<br/>CONCLUSIONS:<br/>This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.

Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Optimisation and scaling of interfacial GeO2 layers for high-k gate stacks on germanium and extraction of dielectric constant of GeO2

<p>Germanium is an attractive channel material for MOSFETs because of its higher mobility than silicon. In this paper, GeO2 has been investigated as an interfacial layer for high-kappa gate stacks on germanium. Thermally grown GeO2 layers have been prepared at 550 degrees C to minimise GeO volatilisation. GeO2 growth has been performed in both pure O-2 ambient and O-2 diluted with N-2. GeO2 thickness has been scaled down to approximately 3 nm. MOS capacitors have been fabricated using different GeO2 thicknesses with a standard high-kappa dielectric on top. Electrical properties and thermal stability have been tested up to at least 350 degrees C. The K value of GeO2 was experimentally determined to be 4.5. Interface state densities (D-it) of less than 10(12) CM-2 eV(-1) have been extracted for all devices using the conductance method.</p>

First-order characteristics of the person-to-vehicle channel at 5.8 GHz

In this paper we investigate the first order characteristics of the radio channel between a moving vehicle and a stationary person positioned by the side of a road at 5.8 GHz. The experiments considered a transmitter positioned at different locations on both the body and receivers positioned on the vehicle. The transmitter was alternated between positions on the central chest region, back and the wrist (facing the roadside) of the body, with the receivers placed on the outside roof, the outside rear window and the inside dashboard of the vehicle. The Rice fading model was applied to the measurement data to assess its suitability for characterizing this emerging type of wireless channel. The Ricean K factors calculated from the data suggest that a significant dominant component existed in the majority of the channels considered in this study.